The rod outer segment (OS) is a modified cilium containing ~1,000 stacked disc membranes that are densely packed with the visual pigment rhodopsin. The mammalian OS is renewed every 10 days;new discs are assembled at the bases of the OS by a poorly understood mechanism. The inner segment, where the biosynthetic and the endocytic pathways are housed, is linked with the OS via the axonemal connecting cilium. The precise route taken by rhodopsin from its site of synthesis to the disc membrane and the mechanism underlying disc renewal are not completely elucidated. However, these questions are not only biologically interesting, but are also clinically relevant because a large number of retinal degenerative diseases are manifested by rhodopsin mislocalization and disc disorganization. Our previous studies suggested that in mammalian rods the new discs are assembled and "grown" via Smad anchor for receptor activation (SARA)-mediated fusions between axonemal rhodopsin vesicles and nascent discs. The new concept that rhodopsin vesicles provide "building blocks" for disc membranes raises many questions. What is the nature of the vesicles? What are the molecular and regulatory pathways that underlie the production and the delivery of these vesicles? Because SARA is an early/sorting endosomal protein that directly binds to rhodopsin and the biosynthetic pathways of most apical surface proteins interact with the endocytic pathway, we will test the hypothesis that post-Golgi rhodopsins traverse the endocytic compartments en route to the OS (Specific Aim 1). How rhodopsin's targeting is affected by strategies interfering with either the endocytic trafficking process or the functions of specific endosomal compartments will be examined. Furthermore, we will test the hypothesis that SARA also has a role in regulating the vesicular trafficking of rhodopsin in addition to its role in the disc fusion (Specific Aim 2). A number of mouse models in which sara gene can be deleted in rods under tissue-specific and/or temporally-regulated fashion will be employed in these studies. Finally, we will test our hypothesis that environmental lighting plays an important role in regulating the OS delivery, and, hence, disc incorporation of rhodopsin (Specific Aim 3). Transfected rodent rods, conditional knockout mice, cell culture models, and several innovative techniques will be employed to comprehensively investigate these questions. PUBLIC HEALTH RELEVANCE: A significant number of genetic disorders affecting the retina alter the membrane trafficking of rhodopsin and the morphogenesis and/or renewal of the light sensing organelle of the rod photoreceptors, outer segment. This fact underscores the importance of our need for a fuller understanding of rhodopsin transport and its involvement in outer segment formation and maintenance. The long-term goal our lab is to unravel the specific steps in rhodopsin trafficking and the identities of the molecules that mediate these processes. In this application, we propose to use a number of highly innovated techniques and a combination of in vitro and in vivo models to tackle these questions. Successful achievement of the proposed specific aims will significantly further our insights into the molecular basis of the vectorial transport of rhodopsin, the genesis and maintenance of the polarity of visual cells. These topics are of central interest in the retinal cell biology. Finally, these studies are highly relevant for our understanding of the etiology of various degenerative retinal diseases and have important implications for future rational therapies for the diseased retina.